Stimulation of γ-Aminobutyric Acid Production in Vine-Ripe Tomato

Aug 1, 2008 - Stimulation of γ-aminobutyric acid (GABA) production under low O2 and high CO2 ... ment effects (2), as well as antihypertensive and na...
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J. Agric. Food Chem. 2008, 56, 7189–7193

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Stimulation of γ-Aminobutyric Acid Production in Vine-Ripe Tomato (Lycopersicon esculentum Mill.) Fruits under Modified Atmospheres YOSHIO MAKINO,*,† NORIKAZU SOGA,† SEIICHI OSHITA,† YOSHINORI KAWAGOE,† § AND ATSUSHI TANAKA Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan, and P-Plus Project, Sumitomo Bakelite Company, Ltd., 495 Akiba-cho, Totsuka-ku, Yokohama 245-0052, Japan

Stimulation of γ-aminobutyric acid (GABA) production under low O2 and high CO2 conditions (adjusted aerobic atmosphere) under which ethanol fermentation could be avoided was studied. Vine-ripe tomato fruits were stored under hypoxia conditions and adjusted aerobic atmospheres as well as in the air at 15 °C for 13 days and at 30 °C for 6 days. At 30 °C tomato fruit GABA concentration under the adjusted aerobic atmosphere (O2 11%, CO2 9%) was significantly higher by 48% than that in air after 6 days from the start of storage. Increased accumulation of alanine under the adjusted aerobic atmosphere supports the observation that this atmosphere stimulates GABA production. The results demonstrate that the concentration of GABA as a beneficial substance for antihypertensive effects and so on can be increased by storing tomato fruits under adjusted aerobic atmospheres for the first time. KEYWORDS: Tomato; Lycopersicon esculentum Mill.; γ-aminobutyric acid; modified atmosphere packaging

INTRODUCTION

γ-Aminobutyric acid (GABA) is an inhibitory amino acid that is widely distributed throughout the biological world (1). It has been reported to have relaxation and immunity enhancement effects (2), as well as antihypertensive and natriuretic effects (3). Therefore, accumulation of GABA in several food types, including rice (Oryza satiVa L.) kernel (4), tea [Camellia sinensis (L.) O. Kuntze] (5), and soybean [Glycine max (L.) Merrill] (6) has been studied. GABA is mainly produced by glutamate decarboxylase (GAD, EC 4.1.1.15) reaction catalyzing decarboxylation from glutamate (7). This suggests that production of GABA is promoted in organisms with a high concentration of glutamate as a main substrate of the enzymatic reaction. In the current study, tomato (Lycopersicon esculentum Mill.) fruits were selected for stimulating GABA production because they contain high glutamate levels (8). Previous results have shown that high levels of GABA accumulate rapidly in plant tissues exposed to a variety of different stresses (1). Stress from anoxia is reported to be effective for stimulating the production of GABA (1); however, horticultural products stored under anoxic/hypoxic * Author to whom correspondence should be addressed (telephone +81 3 5841 5361; fax +81 3 5841 8174; e-mail amakino@ mail.ecc.u-tokyo.ac.jp). † The University of Tokyo. § Sumitomo Bakelite Co., Ltd.

conditions generate off-odors during ethanol fermentation and lose their commercial value (9). The objective of this study was to accumulate GABA by storing tomato fruits using modified atmosphere packaging (MAP), a technique used to prolong the shelf life of horticultural products, in which ethanol fermentation could be avoided (9). MATERIALS AND METHODS Materials. Horticultural Product. Vine-ripe tomato fruits (L. esculentum Mill., Kagome original brand, Round 1 cultivar) harvested November 23, 2006, at Iwaki Onahama Vegetable Farm Ltd. (Fukushima prefecture, Japan) were sampled. After the harvest, the samples were maintained at 10-16 °C and were transported to the laboratory within about 24 h and stored in a constant-temperature unit (15 °C) for another 24 h before being used. The average mass of the 114 pieces of fruit was 0.110 kg (SD ( 0.009; range ) 0.092-0.133 kg). Intact fruits without any pretreatments were used for the following experiments. Pouches. Three-layer laminated [polyethylene terephthalate (PET)/ aluminum/polyethlene] high-barrier pouches Lamizip (AS ONE Corp., Tokyo, Japan) (O2 permeance ) 1.7 × 10-5 mmol · m-2 · h-1 · kPa-1, water vapor transmission rate ) 1.0 mmol · m-2 · h-1, film thickness ) 110 µm; surface area ) 0.056 m2) were used to create hypoxic conditions with high CO2 levels around tomato fruits. Microperforated PET/low-density polyethylene (LDPE) film pouches (O2 permeance ) 0.1 mmol · m-2 · h-1 · kPa-1, water vapor transmission rate ) 10.0 mmol · m-2 · h-1, film thickness ) 47 µm; surface area ) 0.0588 m2) and microperforated polypropylene film pouches (O2 permeance ) 0.25 mmol · m-2 · h-1 · kPa-1, water vapor transmission rate ) 12.5

10.1021/jf801516e CCC: $40.75  2008 American Chemical Society Published on Web 08/01/2008

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Table 1. Mean Values and Standard Deviations (SD) of Tomato Fruitsa in Each of the Storage Methods storage time b,c

0 days storage temperature (°C)

pouch

15

macro-perforated three-layer laminated high barrier microperforated

108.8 108.8 108.8

30

macro-perforated three-layer laminated high barrier microperforated

108.8 108.8 108.8

a

(n ) 6) 5 days or 2 days (n ) 6) 9 daysb or 4 daysc (n ) 6) 13 daysb or 6 daysc (n ) 6) b

mean (g) SD (g)

c

mean (g)

SD (g)

mean (g)

SD (g)

mean (g)

SD (g)

9.7 9.7 9.7

106.6 107.1 111.7

5.9 7.3 8.7

110.5 109.3 108.3

10.9 7.2 5.5

110.7 108.4 107.4

14.3 10.0 8.1

9.7 9.7 9.7

105.2 113.7 119.4

7.6 10.2 5.7

113.8 109.4 111.4

10.4 7.1 4.9

106.5 111.9 109.7

4.8 10.0 10.9

There was no significant difference (P < 0.05, Tukey’s honestly significant difference test) among all mean values. b Storage days at 15 °C. c Storage days at 30 °C.

mmol · m-2 · h-1, film thickness ) 25 µm; surface area ) 0.0588 m2) were used to contain an aerobic controlled atmosphere (O2 11% and CO2 9%) available for storage of tomato fruits (10) at 15 and 30 °C, respectively. The atmospheric conditions were created as described previously (11). Macroperforated pouches (the same PET/LDPE film as the microperforated pouch with 5 mm o.d. × eight holes) were used as a control in which the in-package atmosphere was expected to be the same as the ambient air. Methods. Storage of Tomato Fruits. Two tomato fruits were sealed in a pouch containing 10 g of C2H4 absorbent (12) using an impulse sealer. Fifty-four pouches including C2H4 absorbent were prepared and stored at 15 °C for 13 days and at 30 °C for 6 days (Table 1) in dark constant-temperature units. The storage temperature of 15 °C was selected as a level effective for the keeping quality of tomato fruits according to Nakhasi et al. (13) On the other hand, the storage temperature of 30 °C was selected to stimulate enzymatic reactions associated with GABA production. Storage periods at each temperature were determined subjectively with storage terminated when the fruit visually lost its commercial value in air caused by softening (14) or fungi (15). It was presumed that differences of relative humidity among the pouches did not affect the stimulation of GABA production because the humidity was kept at a level which could not stimulate GABA production caused by water stress (16). Analysis of In-Package Atmospheres. Aliquots (300 µL) of inpackage gas were sampled over time using a gastight syringe inserted through a silicone rubber septum stuck on the pouch. O2, CO2, and C2H4 concentrations in the sampled gas were measured by gas chromatography as reported by Makino et al. (11, 17) Analysis of Free Amino Acid Concentrations in Tomato Fruits. All tomato samples were analyzed for free amino acid concentrations. A hull-less fruit, cooled with crushed ice, was homogenized, and the protein in an aliquot (0.5 g) of the homogenate was denatured by adding 0.5 mL of trichloroacetic acid. The mixed solution was centrifuged at 2300g for 10 min. An aliquot (0.04 mL) of supernatant was diluted by 0.96 mL of 0.02 M HCl (pH 2.2) and filtered using a membrane filter (0.2 µm pore size). Concentrations of free amino acids in the treated samples were analyzed using a JLC-500/V2 Amino Acid Analyzer (Japan Electron Optics Laboratory Ltd., Tokyo, Japan) at the Chemical Analysis Division of the Research Facility Center for Science and Technology of the University of Tsukuba (Tsukuba, Ibaraki, Japan). Amino acid concentrations were expressed on the basis of fresh weight. Statistical Analysis. JMP7.0.1 software (SAS Institute Inc., Cary, NC) was used for statistical analysis. When between-class variation was significant at P < 0.05 using one-way analysis of variance (ANOVA) of the data, mean values were compared by the least significant difference (LSD) test (P < 0.05). RESULTS

Gas composition in the macroperforated pouch was the same as that in the surrounding air regardless of temperature. Changes in O2 and CO2 concentrations in the high-barrier and microperforated film pouches over time are shown in Figure 1. In the high-barrier pouch at 15 °C, the mean O2 concentration decreased from approximately 21 to 3.4% after 120 h of storage

Figure 1. In-package atmosphere changes in pouches at 15 and 30 °C (n ) 3). Open and solid symbols denote microperforated film and threelayer laminated high barrier pouches, respectively. Circles and triangles denote in-package O2 and CO2 concentrations, respectively. Mean ( SE of three observations has been plotted. In-package O2 and CO2 concentrations were significantly different (P < 0.05; one-way ANOVA with LSD test) among pouches in each temperature and in the same time interval.

and remained at about 5% until the end of storage (312 h). The mean CO2 concentration increased from 0 to 12% after 46 h and gradually increased to 19% at the end of storage. At 30 °C, the mean O2 concentration decreased to 3.4% after 20 h from the start of storage and remained at about 2% until the end of storage (144 h). The CO2 concentration increased to 18% after 22 h of storage and continued to increase until reaching 41% at the end of storage. In the microperforated film pouches at 15 °C, a steady state was reached after 50 h from the start of storage. Mean O2 and CO2 concentrations of 12 and 8%, respectively, were maintained until the end of storage (312 h). At 30 °C, a steady state was reached after 20 h, and mean O2

Aerobic Increase of GABA in Tomatoes

Figure 2. Changes in γ-aminobutyric acid in tomato fruit at 15 and 30

°C (n ) 6). Circles, triangles, and squares denote values under ambient air, hypoxic conditions with high CO2 levels, and adjusted aerobic (low O2 and high CO2 conditions) atmospheres, respectively. Mean ( SE of six observations has been plotted. Within the same time interval, symbols followed by the same letter are not significantly different (P < 0.05; oneway ANOVA with LSD test).

and CO2 concentrations of 11 and 9%, respectively, were maintained until the end of storage (144 h). The gas composition in the storage packaging was, regardless of storage temperatures, hypoxic in the high-barrier pouch and aerobic in the microperforated film pouch. C2H4 concentrations were kept at 0 ppm in all of the pouches. Therefore, in-package C2H4 did not affect the experimental data. Changes in GABA concentrations over time are shown in Figure 2. At 30 °C, the GABA concentration was significantly higher in the high-barrier pouch than in the macroperforated pouch after 96 h of storage. In addition, GABA concentrations were significantly higher in both the high-barrier and microperforated film pouches than in the macroperforated pouches after 144 h. At 15 °C, no significant differences were observed among the three packaging methods. As the experiment durations differed for the 15 and 30 °C samples, the time available to measure GABA was markedly different. However, when GABA concentrations after 144 h at 30 °C, after 120 h at 15 °C, and after 216 h at 15 °C are compared, the influence of higher temperature on the stimulation of GABA production is apparent. On the other hand, even under hypoxic conditions with high CO2 levels, GABA production was not stimulated at 15 °C. A decrease in storage O2 and an increase in storage CO2 concentrations are considered to promote GABA production, and our results indicate that the effect is influenced by storage temperature.

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Figure 3. Changes in glutamate in tomato fruit at 15 and 30 °C (n ) 6). Circles, triangles, and squares denote values under ambient air, hypoxic conditions with high CO2 levels, and adjusted aerobic (low O2 and high CO2 conditions) atmospheres, respectively. Mean ( SE of six observations has been plotted. Within the same time interval, symbols followed by the same letter are not significantly different (P > 0.05; one-way ANOVA with LSD test).

In the perforated pouches, glutamate concentrations were significantly higher at 216 h of storage at 15 °C (Figure 3). No other significant differences were observed among the different packaging pouches at either storage temperature. After 96 h from the start of storage at 30 °C, the alanine concentration in the microperforated film pouch became significantly higher compared with other pouch types and reached 1134 mmol · g-1 of FW at the end of storage (144 h) (Figure 4). This level was 2.2 times the value at the end of storage (312 h) at 15 °C. Thus, the alanine concentration was highest under the adjusted aerobic atmosphere, and its concentration was affected by storage temperature. DISCUSSION

Among the atmospheres tested in the current study, production of GABA in tomato fruit was most stimulated under the hypoxic condition created by the high-barrier pouches (Figure 2), similar to the observations in a study on tea (5). Under the adjusted aerobic atmospheres created in the current study, tomato fruits respired without ethanol fermentation as reported by Geeson et al. (10). Although no reports were found concerning stimulation of GABA production by weak O2 stress under aerobic conditions, we observed that GABA concentration in the adjusted aerobic atmosphere was significantly higher than that observed in ambient air at 30 °C. Kinnersley and Turano (1) reported that the GAD reaction is stimulated by the acidification of plant tissue. GABA is predominantly produced by the GAD reaction,

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Figure 4. Changes in alanine in tomato fruit at 15 and 30 °C (n ) 6).

Circles, triangles, and squares denote values under ambient air, hypoxic conditions with high CO2 levels, and adjusted aerobic (low O2 and high CO2 conditions) atmospheres, respectively. Mean ( SE of six observations has been plotted. Within the same time interval, symbols followed by the same letter are not significantly different (P > 0.05; one-way ANOVA with LSD test).

which is an irreversible reaction involving consumption of H+. An accumulation of H+ reduces the pH and stimulates the GAD reaction. Reduction of O2 concentration around tomato fruits might result in decreased oxidation of H+ on an active site of cytochrome c oxidase as the terminal enzyme in the respiratory chain (18). Glutamate is a main substrate in the GAD reaction, which suggests that the GAD reaction may be stimulated with an increase of glutamate. This is supported by a study using rice kernel (4). If glutamate is consumed to produce GABA, the concentration of glutamate would decrease with GABA accumulation, as has been demonstrated using radish leaves (19). However, the relationship between the concentrations of GABA and glutamate in the current study remains unclear (Figures 2 and 3). Glutamate is produced from R-ketoglutarate by glutamate dehydrogenase (GDH, EC 1.4.1.2) and GABA transaminase (GABA-T, EC 2.6.1.19) reactions as well as being consumed by the GAD reaction (7). The obscurity of this relationship may be related to the complex metabolic pathways involved. Streeter and Thompson (19) demonstrated that glutamate might be provided by decomposition of proteins in the case of long-term incubation in their paper. Therefore, they conducted incubation of trimmed radish leaves for 6 h at room temperature to detect a decrease in glutamate with an increase in GABA. In the current study, intact tomato fruits were stored for 6 days at 30 °C and for 13 days at 15 °C. Liu and Luh (8) reported that concentra-

Makino et al. tions of GABA and glutamate in tomato paste from harvestdelayed fruit were elevated with the delay of harvest. Glutamate may be increased by the reaction of nitrogen fixation, known as the glutamine synthetase (GS, EC 6.3.1.2)-glutamate synthase (GOGAT, EC 1.4.1.13) cycle, during cultivation. Differences in sample storage conditions appear to be the cause of the differences in the glutamate data mentioned above. Streeter and Thompson (19) reported that alanine and GABA were accumulated in radish leaves under anoxic/hypoxic conditions. In the current study, alanine accumulated the most under the adjusted aerobic atmospheres (Figure 4). Miyashita et al. (20) reported that O2 stress promoted the decomposition of accumulated alanine by alanine aminotransferase (AlaAT, EC 2.6.1.2) in Arabidopsis [Arabidopsis thaliana (L.) Heynh.]. This suggests that alanine may be decomposed under hypoxic conditions through strong O2 stress and be maintained under adjusted aerobic atmospheres through weak O2 stress. Accumulation of alanine in tomato fruits under the adjusted aerobic atmosphere supports the observation that atmospheric conditions stimulate the production of GABA (19). The results demonstrate that GABA production can be stimulated by storing tomato fruit under an adjusted aerobic atmosphere in which ethanol fermentation can be avoided. In general, freshness is an important quality of horticultural products, which is known to degrade with postharvest storage time. Cold storage, controlled atmosphere, MAP, and 1-methylcyclopropene are conventional techniques applied to slow the deterioration of horticultural products. In this paper, an unconventional approach to increase the concentration of GABA as a beneficial substance for maintaining postharvest horticultural product quality using MAP was demonstrated. According to the research reported by Inoue et al. (21), a dose (10-12 mg) of GABA for 12 weeks to people with mild hypertension was effective for lowering their systolic and diastolic blood pressures by 17.4 ( 4.3 and 7.2 ( 5.7 mmHg. The dose is equivalent to one-fifth of a fruit stored under adjusted aerobic atmosphere created by MAP or to one-third of a fruit stored in the air at 30 °C for 6 days in the current study. This suggests that MAP is useful for increasing the antihypertensive effect of tomato fruits. However, the temperature (30 °C) effective for stimulating GABA production may be unsuitable for the storage of tomato fruits. Therefore, other temperatures within the range of 15-30 °C, which may be effective for stimulating GABA production and maintaining product quality, should be researched. GABA production in vine-ripe tomato fruits under low O2 and high CO2 conditions (adjusted aerobic atmosphere) was stimulated significantly more than in air at 30 °C. Accumulation of alanine under the adjusted aerobic atmosphere supports the observation that this atmosphere type is effective for stimulating GABA production. However, the relationship between glutamate, a main enzymatic substrate of GAD, and GABA concentrations remains unclear because of the complex metabolic pathways in the production of glutamate. ACKNOWLEDGMENT

We are grateful to Kagome Co. Ltd. (Tokyo) for donating the tomato fruits. LITERATURE CITED (1) Kinnersley, A. M.; Turano, F. J. γ-Aminobutyric acid (GABA) and plant responses to stress. Crit. ReV. Plant Sci. 2000, 19, 479– 509. (2) Abdou, A. M.; Higashiguchi, S.; Horie, K.; Kim, M.; Hatta, H.; Yokogoshi, H. Relaxation and immunity enhancement effects of

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(13) Nakhasi, S.; Schlimme, D.; Solomos, T. Storage potential of tomatoes harvested at the breaker stage using modified atmosphere packaging. J. Food Sci. 1991, 56, 55–59. (14) Saladie´, M.; Matas, A. J.; Isaacson, T.; Jenks, M. A.; Goodwin, S. M.; Niklas, K. J.; Xiaolin, R.; Labavitch, J. M.; Shackel, K. A.; Fernie, A. R.; Lytovchenko, A.; O’Neill, M. A.; Watkins, C. B.; Rose, J. K. C. A reevaluation of the key factors that influence tomato fruit softening and integrity. Plant Physiol. 2007, 144, 1012–1028. (15) Oladiran, A. O.; Iwu, L. N. Studies on the fungi associated with tomato fruit rots and effects of environment on storage. Mycopathologia 1993, 121, 157–161. (16) Thompson, J. F.; Stewart, C. R.; Morris, C. J. Changes in amino acid content of excised leaves during incubation I. The effect of water content of leaves and atmospheric oxygen level. Plant Physiol. 1966, 41, 1578–1584. (17) Makino, Y.; Ichimura, M.; Kawagoe, Y.; Oshita, S. Cytochrome c oxidase as a cause of variation in oxygen uptake rates among vegetables. J. Am. Soc. Hortic. Sci. 2007, 132, 239–245. (18) Makino, Y.; Iwasaki, K.; Hirata, T. Oxygen consumption model for fresh produce on the basis of adsorption theory. Trans. ASAE 1996, 39, 1067–1073. (19) Streeter, J. G.; Thompson, J. F. Anaerobic accumulation of γ-aminobutyric acid and alanine in radish leaves (Raphanus satiVus L.). Plant Physiol. 1972, 49, 572–578. (20) Miyashita, Y.; Dolferus, R.; Ismond, K. P.; Good, A. G. Alanine aminotransferase catalyses the breakdown of alanine after hypoxia in Arabidopsis thaliana. Plant J. 2007, 49, 1108–1121. (21) Inoue, K.; Shirai, T.; Ochiai, H.; Kasao, M.; Hayakawa, K.; Kimura, M.; Sansawa, H. Blood-pressure-lowering effect of a novel fermented milk containing γ-aminobutyric acid (GABA) in mild hypertensives. Eur. J. Clin. Nutr. 2003, 57, 490–495. Received for review May 15, 2008. Revised manuscript received June 29, 2008. Accepted June 30, 2008. This study was financially supported by a Grant-in-Aid for Scientific Research (No. 19380140) from the Japan Society for the Promotion of Science.

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